Recently, immuno-cell therapy is focused as a new therapy for intractable diseases including cancer. The immuno-cell therapy is to collect and activate patient's own immunocytes, particularly white blood cells before returning them to the patient to artificially enhance the immunizing power. This therapy has the advantage of reduced side effects compared to conventional anticancer drug treatment and the like.
As the immuno-cell therapy, activated autologous lymphocyte therapy is already becoming available which involves antigen-non-specifically activating lymphocytes in vitro with a lymphokine and then returning them into the body; however, there is need for the availability of therapy using cytotoxic T lymphocytes (hereinafter referred to as CTLs) which have stronger cytotoxic activity and specifically recognize and injure a lesion.
Here, as currently attempted therapy using CTLs, a method is adopted, for example, which involves directly administering a cancer antigen peptide or the like to a patient; however, in this case, the ability thereof to induce CTLs admits of improvement because the patient often has lowered immunity. Accordingly, there is dendritic cell vaccine therapy or the like which involves contacting an antigen with antigen presentation cells, for example, dendritic cells (hereinafter also referred to as DCs), or the like and thereby strongly causing the cells to present the antigen to induce disease antigen specific CTLs by the DCs in the body.
Some current typical methods for inducing disease antigen specific CTLs are shown below as examples.
For example, in Non Patent Literatures 1 and 2, 9-mer peptides such as an epitope for a particular disease antigen are added to peripheral blood mononuclear cells to attempt the induction of CTLs from there. In addition, in Non Patent Literatures 3 and 4, DCs are obtained from peripheral blood mononuclear cells and an antigen peptide is added thereto to impart an antigen-presenting function; CD8-positive T cells separated from peripheral blood lymphocytes are cultured together with the resultant DCs to induce CTLs specific for the disease antigen.
Further, in Non Patent Literature 5, DC vaccination is carried out using DCs obtained from hematopoietic precursor cells and an antigen peptide and then monocyte-derived DCs to which the antigen is added are co-cultured with CD8-positive T cells separated from peripheral blood mononuclear cells to induce CTLs specific for the antigen.
As can be seen from their use in the above examples, dendritic cells are high in antigen-presenting capability and in the ability to induce disease antigen specific CTLs among other antigen-presenting cells; thus, technological development for obtaining them is under way.
However, to obtain dendritic cells for dendritic cell vaccination, it is typically necessary to collect human peripheral blood, separate cells called monocytes from there, and culture them after adding IL-4, GM-CSF and the like. This process is cumbersome and at present has a problem that the number and function of cultured cells vary depending on the skill of a cell culture technician.
γδT cells, which are activated by a non-peptide antigen, are cells responsible for natural immunity; these cells have recently been found to exert MHC-unrestricted cytotoxic activity (non-specific activity) on cancer cells. Thus, immunotherapy using the strong anti-tumor activity of these γδT cells is investigated.
Because γδT cells are activated by recognizing a non-peptide antigen, they can be stimulated, for example, with an alkylamine or a bisphosphonate as the non-peptide antigen for activation and/or proliferation; γδT cells separated from peripheral blood have been caused to recognize the non-peptide antigen in vitro to perform studies on the activation and/or proliferation thereof (for example, Non Patent Literature 6).
However, a problem of γδT cells, which are generally present in an amount of only 1 to 5% in peripheral blood, is that the purity and number of the γδT cells sufficient for medical treatment cannot be secured even if a small amount of blood is collected and then the cells therefrom are activated and/or proliferated. Increasing the amount of blood collection from a patient to secure the purity and number of the γδT cells sufficient for medical treatment also poses a problem that it imposes a great burden on the patient.
Patent Literature 1 also discloses a method which involves adding a bisphosphonate to peripheral blood mononuclear cells to activate and/or proliferate γδT cells.
Thus the culture and use of more effective cells are very important for immuno-cell therapy; the development of a wide variety of methods and techniques are still carried out.
In addition, one type of cells are mostly cultured under present conditions and used for medical treatment in immuno-cell therapy; however, various cells may probably be mixed and used for medical treatment, given such characteristics that as described above, for example, disease antigen specific CTLs attack targeted cells or tissue in an MHC-restricted manner, for example, specifically for an antigen presented in MHC class I and γδT cells attack cells or tissue in an MHC-unrestricted manner.
However, to culture a plurality of types of immune cells for medical treatment, culture steps had to be concurrently carried out for a number of the types in practice. Culture conditions suitable for each type of cells are different from those for the other; if a conventional culture method suited for one type of cells is used, it will be very difficult, for example, to simultaneously and efficiently culture sufficient numbers of disease antigen specific CTLs and γδT cells for exerting a therapeutic effect; and such effective culture and induction methods are not yet brought to perfection.